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Semin Nephrol. Author manuscript; available in PMC 2019 March 01.
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Published in final edited form as:

Semin Nephrol. 2018 March ; 38(2): 193–205. doi:10.1016/j.semnephrol.2018.01.008.

Contributory Role of Gut Microbiota and Their Metabolites

Towards Cardiovascular Complications in Chronic Kidney
Disease
Daniel Y. Li and W. H. Wilson Tang, MD
Cleveland Clinic Lerner College of Medicine at Case Western Reserve University; and Center for
Clinical Genomics, Cleveland Clinic; Department of Cardiovascular Medicine, Heart and Vascular
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Institute, Cleveland Clinic

Abstract
The gut microbiome has recently emerged as a novel risk factor that impacts health and disease.
Our gut microbiota can function as an endocrine organ through its unique ability to metabolize
various dietary precursors, and can fuel the systemic inflammation observed in chronic disease.
This is especially important in the setting of chronic kidney disease, where microbial metabolism
can directly contribute to accumulation of circulating toxins that can then alter and shift the
balance of microbiota composition and downstream functions. To study this process, advances
inomics technologies are providing opportunities to understand not only the taxonomy but also
functional diversity of our microbiome. We can also reliably quantify en masse a wide range of
uremic byproducts of microbial metabolism. Herein, we examine the bidirectional relationship
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between the gut microbiome and the failing kidneys. We will describe potential approaches
targeting gut microbiota for cardiovascular risk reduction in chroinc kidney disease using an
illustrative example of a novel gut-generated metabolite, trimethylamine N-oxide (TMAO).

Introduction
In the 1800s the English physician Richard Bright established a relationship between
chronic kidney disease (CKD) and the clinical abnormalities brought about in the uremic
state or the “residual syndrome” including cardiovascular disease (CVD)1, 2. It has long been
postulated that such increased CVD risk in CKD patients is partly due to a shared set of
traditional risk factors such as diabetes, hypertension, albuminuria, dyslipidemia, and
smoking3. However, such traditional risk factors do not entirely explain the accelerated
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progression of CVD in CKD patients. Since the large majority of morbidity and mortality of
CKD remains CVD-related, elucidation of novel CVD risk factors may improve clinical
outcomes of CKD patients.

Address for Correspondence: W. H. Wilson Tang, MD, 9500 Euclid Avenue, Desk J3-4, Cleveland, OH 44195. Phone: (216)
444-2121 / Fax: (216) 445-6165 / tangw@ccf.org.
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The contribution of specific gut microbiota generated metabolites towards uremia and poor
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outcomes have been documented in observational studies and associated with important
clinical endpoints such as progression of renal failure, CVD events, and mortality4, 5. Today,
the perception of our microbiome has evolved from merely an inert set of microorganisms to
a bona fide “endocrine organ.” Weighing in at approximately 2 kilograms, our intestinal
microflora plays a vital role to maintain a symbiotic relationship with its host6.

Multiple efforts such as the Human Microbiome Project and MetaHIT are cataloging the
human microbiome7, 8. At birth, the gut microbiome is relatively sterile. However,
continuous exposure to the environment results in subsequent colonization of trillions of
bacteria (> 1014)9, 10. This composition of bacteria is now believed to be a sensitive factor
that alters our risk for disease. In the setting of CKD, a comparison of the gut microbiota
composition between those with end-stage renal disease (ESRD) and healthy individuals
demonstrated the increased relative abundance of at least 190 bacterial operational
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taxonomic units (OTU) 11. The authors then extended their findings to a model of 5/6
nephrectomized rats which showed that a decrease in renal function also resulted in a
distinct bacterial community11. In addition, the mode of dialysis can also impact the gut
microbiota composition12. Based on these “environmental exposures,” our gut microbiome
can generate products that are dependent on renal clearance that are either beneficial and/or
detrimental to our health. In fact, production of uremic toxins may involve several different
sources, ranging from the body itself (without intestinal processing), unmodified absorption
of dietary products (such as advanced end glycation products), to metabolites that are
produced in the intestine from precursors originating from microbial fermentation13 – all
accummulating in the setting of impaired renal clearance.

How Uremic Solutes and Metabolites Impact the Microbiota

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Dysbiosis, or imbalance of gut microbial composition, can stem from many processes
including an altered diet as well as colonic compensation for increased uremic toxins.
Specifically, uremic metabolites and solutes are produced with contributions at many steps
including direct microbiota transformation, intestinal wall conjugation, liver conjugation or
endogenous generation (Figure 1). As the glomerular filtration and compensatory tubular
secretion become impaired in CKD and tubular injury, increased nitrogen compounds such
as urea and uric acid may instead excreted via the intestines. As an example, increased urea
secretion through the gastrointestinal tract is converted to ammonium which then leads to
elevated intraluminal pH as well as mucosal damage14. This influx of nitrogen-rich
compounds, along with the damage incurred by its metabolic byproducts, can lead to further
gut dysbiosis. In the setting of elevated uremic toxins, bacterial species that contain enzymes
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capable of metabolizing nitrogen compounds such as p-cresol and indole, urea, and uric acid
as sources for fermentation will out-proliferate bacteria such as Lactobacillae or groups that
utilize dietary fiber to convert into short chain fatty acids (SCFA)15. These functional
changes have been reported by Wong et al. in which 19 families of bacteria containing
urease, uricase, and indole or p-cresol-forming enzymes were identified predominantly in
the setting of ESRD15, while there were decreased bacterial families possessing SCFA
butyrate-forming enzymes. Many other external factors can also influence gut microbial
composition. For example, patients with CKD maintains a difficult balance between

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adequate nutrition to promote symbiosis while avoiding potassium and phosphorus

overload16. Physiologic factors such as gut edema and reduced intestinal transit will
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influence the intestinal microflora. Patients with CKD often require chronic medications
including phosphate binding agents, iron supplements as well as antibiotics that can directly
or indirectly influence gut microbial composition.

The Microbiota as a Driver of Persistent Inflammation in CKD

In a healthy individual, the mucosal lining of the gut epithelium consists of the mucus layer
as well as protective factors such as defensins and lectins that can create a barrier between
the epithelium and immune system from contact with the microbiota17, 18. However, in
autopsy studies of hemodialysis patients from the 1980s, chronic inflammation was noted
throughout the gastrointestinal tract beginning from the esophagus and ending at the colon19.
The source of inflammation was attributed to factors such as the dialysis interface,
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subclinical infection, iron administration or pre-existing cardiac dysfunction20. Further

investigation of intestinal permeability in rats showed that molecular markers such as
polyethylene glycol of varying molecular weight were detectable in rats with CKD21.
Structurally, the intestinal epithelium consists of claudins, occludin, E-cadherin and zona
occludens which help protect the epithelial junctions22. Studies targeting these epithelial
factors confirmed a depletion of the tight junction proteins such as claudins, occludin-1, and
zona occludens-123. This increase in permeability is hypothesized to allow for a “leaky gut”
and thus contribute to chronic endotoxemia as well as the increased passage of uremic
solutes from the gut into the body24. Sequencing of the collecting lymph nodes in the
mesentery demonstrated that that colonic bacteria could also be detected25. The presence of
microbes that pass through the gut barrier can further induce several families of pattern
recognition receptors including the NLRP3 (nucleotide-binding oligomerization domain
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[NOD]-like receptor family, pyrin domain containing 3) inflammasome, toll-like receptors,

and NOD-like receptors26. Once tight junction breakdown occurs through multiple means,
experimental evidence shows that increased levels of urea during decreased clearance results
in compensatory excretion through the gut27. Accumulation of urea in the gut lumen results
in a different nutritional source that is utilized by gut bacteria that contain urease. This result
in the production of ammonia which is then hydrolyzed to ammonium hydroxide, which can
injure the gut epithelial barrier14, 27. This injury results in recruitment of inflammatory cells
such as leukocytes which then feeds forward into increased levels of cytokines that stimulate
endocytosis of the tight junction proteins.

Direct evidence for intestinal breakdown and subsequent bacterial translocation as a source
of inflammation in CKD was shown by Anders et al. using a Col4a3-knockout mice to
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simulate CKD28. In their model, a co-housing strategy was used to ensure that cage effects
did not bias the results. The uremic mice developed increased inflammation with increased
levels of pentraxin-2, interleukin(IL)-6, and T cells in the spleen. However, IL-1β, another
cytokine well described to influence gut permeability was statistically unchanged. Bacterial
sequencing also showed demonstrable alterations in the gut flora of the Col4a3-knockout
mice. To then confirm a microbiota mediated inflammatory effect, the authors used
antibiotics in the uremic mice and demonstrated decreased inflammatory factors, decreased

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activated T cells, and a recovery of regulatory T cells. More importantly, renal function was
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not changed, which rules out antibiotic effects on the kidney itself.

Targeting inflammatory mediators as a method to reduce CVD has recently been shown in
humans in the Canakinumab Anti-inflammatory Thrombosis Outcomes Study (CANTOS)29.
Blocking IL-1β led to reduced CVD events but those with heart failure, nephropathy or
estimated glomerular filtration rate <30 ml/min/1.73m2 were not included in this study. In
CKD, another avenue of exploration is the nuclear transcription factor Nrf2, which has been
investigated for its anti-oxidant and anti-inflammatory roles30. Lau et al. have shown that
inflammation suppresses Nrf2 expression, but treatment with Nrf2 agonists can result in
improved expression of tight junction proteins as well as improved inflammation31.
However, a phase 3 trial using a Nrf2 agonist, bardoxolone methyl, to delay ESRD
progression surprisingly resulted in increased CVD adverse events32. Hence, further
investigations of strategies that target the instigators or mediators of inflammation in CKD
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are therefore warranted, which prompted the growing interests in gut microbiota.

Evidence for Gut Microbiota Derived Uremic Toxins

In recent years, rapid expansion of “-omics” technology has enabled “high-throughput”
discoveries at every level of biology, which is in stark contrast of a century ago when
phlebotomy and subsequent analysis of urea in the blood and urine was state of the art1. The
contribution of gut microbiota towards the generation of uremic toxins has been investigated
over half a century when gnotobiotic anephric rats were observed to have longer survival
compared to their germ-inhabited counterparts33. As early as in the 1970s, patients with
CKD were found to have significantly altered microbiota34, and that alteration of the
intestinal bacterial flora with antibiotics markedly reduced serum dimethylamine and
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trimethylamine concentrations and modified mental status symptoms35. Furthermore,

removal of the microbiota via antibiotic use also resulted in both the fecal and urinary
excretion of phenolic and aromatic bacterial metabolism products in a pig model36. These
seminal observations established the association between gut microbiota-derived uremic
toxins and CKD disease progression.

Today, integration of metabolomics, proteomics, and microbiomics can accelerate the

discovery of novel retained uremic solutes. For example, combining proteomics and
metabolomics will allow the identification and quantification of proteins, peptides as well as
small metabolites under 1,000 Da. These products represent the phenotypes of both the host
and microbial metabolism. We can then assimilate the findings of the microbiota through
both the taxonomy via 16s sequencing as well as microbial community dynamics by
metagenomic sequencing. Indeed, accumulation of multiple colonic-derived metabolites has
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been identified through untargeted metabolomics in biological samples from dialysis

patients, in which specific metabolites known to be generated by colonic microbiota were
noticeably absent after colectomy while being >20-fold higher without colectomy37.
Moreover, these metabolites were able to distinguish patients with and without colectomy
after hierarchical clustering. This survey allowed identification and confirmation of multiple
colonic-derived metabolites, even though the authors were unable to clarify the identity of
many of these metabolites. A similar approach utilized untargeted metabolomics in the

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plasma produced by intestinal microbiota of rats with normal kidney function, among them
~10% were considerably different between the gnotobiotic and conventionally-raised rats38.
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Amongst the identified discriminators, the authors identified nine indole and phenyl
derivatives, with many overlapping with previous CKD studies from Aranov et al. along
with seven other solutes that were elevated. Of note, these metabolites were also noted to be
substrates of common xenobiotic transporters such as organic anion transporters.
Applications of these retention solutes are further demonstrated in a variety of studies using
multi-marker methodologies to expand the prognostic capabilities in CKD. For example, in
the Framingham cohort, a multi-marker panel of such metabolites could predict the
development of CKD after adjustments for risk factors such as kidney function,
demographics, comorbidities, and proteinuria at baseline39. From their screen of over 200
metabolites, those in the tryptophan, choline, and purine metabolic pathways were highly
associated in the CKD population. They further quantified which metabolites had a higher
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secretion potential by measuring the venous to arterial metabolite ratios in 9 patients as a

surrogate for renal tubular involvement. Another metabolomics screen in stage 3-4 CKD
patients revealed 14 elevated metabolites in those with disease. This screening process
identified several known uremic solutes including p-cresyl sulfate (pCS). Moreover, two
novel metabolites, dimethyl sulfone, and 2-hydroxyisobutyric acid were also found40.

An updated review of uremic retention solutes by the European Uremic Toxin Work Group
has now listed over 150 different compounds41. Given the relative ease for novel small
molecular discovery to associate with the CKD phenotype, it is also imperative for the
pathophysiologic mechanisms of these novel uremic retention solutes to be investigated,
especially in relation to gut microbiota metabolism. Here, we take a closer look at the
beneficial bacterial fermentation product SCFA, the well-studied uremic solutes indoxyl
sulfate (IS) and p-CS), and the more recently discovered trimethylamine N-oxide (TMAO).
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A recent review from Ramezani et al. provides primers on additional uremic solutes42.

Short Chain Fatty Acids

On the beneficial spectrum of microbiota generate metabolites, distal gut anaerobic
fermentation of undigested nutrients such as resistant starches, dietary fiber, various
polysaccharides produce organic fatty acids containing one to six carbons called short-chain
fatty acids (SCFAs). The majority of SCFAs consist of acetate, butyrate, and propionate with
a distribution of 60% acetate and equal 20% amounts of butyrate and proprionate43, 44. The
colonic epithelium efficiently absorbs SCFAs through both passive and active
mechanisms45. The variety of host functions affected by SCFAs has been expanding. The
most abundant SCFA readily detectable in the blood, acetate, is a source of energy for
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peripheral cells46. Similarly, butyrate provides energy for the maintenance of the ceco-
colonic epithelium while propionate is a source for gluconeogenesis in the liver. Together,
these SCFA are estimated to provide between 5 to 10 percent of the energy source in healthy
individuals43.

More than just a source of nutrition, recent studies have suggested that SCFAs also play
important roles in processes, such as regulation of blood pressure and modulation of
systemic inflammation. Increased fruit and vegetable consumption has also been shown to

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result in increased acetate production. This increase in acetate has been demonstrated to
reduce blood pressure while preventing renal and cardiac fibrosis47. Recently, several novel
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receptors such as G-protein coupled receptors 41 and 43 (Gpr41, Gpr43), have been
identified to be targets of SCFA48. However, the methods by which SCFA can alter Gpr43
has been shown to counteract increases in renin secretion by olfactory receptor 78 (olfr78) in
the kidney49. SCFAs plays a major role in inflammation and mediating intestinal tight
junction integrity50. SCFAs have been shown to prevent inflammation through increased
anti-inflammatory cytokine IL-10 and decreased proinflammatory IL-12 and tumor necrosis
factor alpha (TNF-α) secretion50-52. While gnotobiotic mice are more susceptible to
ischemia-reperfusion kidney injury, treatment with SCFAs can protect mice through
decreased inflammation, decreased apoptosis, and increased autophagy53.

Indoxyl Sulfate and P-Cresyl Sulfate, Colon Derived Solutes

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Indoxyl sulfate and its related indole compounds are produced by sulfation in the liver after
degradation of tryptophan by bacterial tryptophanase. Similarly, pCS is a major circulating
metabolite of p-cresol conjugation in the intestinal wall54. p-Cresol is a product of colonic
bacterial fermentation from phenylalanine and tyrosine. Both IS, and pCS are protein-bound
and are not readily filtered by the kidney or removed by hemodialysis5. Instead, it is
dependent upon secretion by organic anion transporters and accumulates during CKD
progression. Niwa described this “protein metabolite” hypothesis in 1997, and because of
this physiology, these metabolites have been the focus of studies in uremia55.

Many clinical studies have examined the association between IS and pCS with both CKD
progression and their potential contribution to CVD. Circulating levels of IS and PCS have
been associated with the severity of CKD. Elevated serum IS has been directly associated
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with aortic calcification, arterial stiffness represented by altered pulse wave velocity,
peripheral vascular disease, and mortality56. Similarly, pCS levels correlated with impaired
renal function and increased CVD events57. Furthermore, a prospective study of 268 patients
with CKD found associations for both IS and pCS with predicated loss of kidney function58,
and a meta-analysis has found associations for both PCS and IS with mortality, although
only pCS (not IS) was associated with CVD mortality59.

A wide array of mechanisms for which IS and PCS can contribute to both CKD and CVD
has been explored and summarized in Table 1. Early investigations showed that IS
administration to rats was capable of stimulating monocyte infiltration with the production
of factors related to tubulointerstitial fibrosis such as transforming growth factor beta (TGF-
β) or tissue inhibitor of metalloproteinases (TIMPs)60. IS has found be mechanistically
related to multiple CVD phenotypes61. Similar to IS, direct administration of PCS promotes
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renal tubular damage in 5/6-nephrectomized rats that were attributed to alterations in

NADPH oxidase activity, reactive oxygen species (ROS) production, and induction of
inflammatory cytokines62. As previously mentioned, pCS and IS are protein bound and not
easily removed via hemodialysis. Therefore, it is important to assess their effects as a
measure of free concentration and not of total solute concentration. A systematic review
analyzed 27 studies in which free concentrations of PCS and IS were studied in cell types
such as the endothelium, hepatocyte, tubular cells, and cardiac tissue5. This rigorous review

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with stringent standards confirms the toxicity of IS and pCS and support their roles in
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vascular and renal disease progression.

Trimethylamine N-oxide, a Meta-organismal-Derived Metabolite

Trimethylamine N-oxide is a product of bacterial metabolism of dietary precursors that
contain the trimethylamine (TMA) moiety such as phosphatidylcholine and carnitine63, 64.
TMA generated by bacterial lyases are efficiently absorbed by the intestine and subsequently
converted to TMAO by liver flavin-containing monooxygenase 3 (FMO3) in the human
host65. Classically, TMAO has been studied for its activity as an organic osmolyte and helps
counterbalance the extreme salinity and osmotic pressures which deep-sea fish encounter66.
This molecule has also been studied for its molecular chaperone properties67, and has long
been thought to be a nitrogenous waste metabolite that is largely excreted by the kidneys.
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Over the past years, TMAO has emerged as a novel contributor to the development of
atherosclerosis, and the scope of TMAO research has rapidly expanded towards other
diseases such as CKD and heart failure4, 68. Wang et al. first identified TMAO through an
untargeted metabolomics screen of patients at high risk for cardiovascular disease, and
confirmed these findings in a large prospective cohort in which elevated fasting levels of
TMAO portend poorer long-term major adverse cardiac events69, 70. In ApoE null mice,
direct TMAO feeding was observed to cause atherosclerosis while antibiotics resulted in
decreased atherosclerotic plaques. These findings established a direct microbiota-dependent
link between dietary nutrients, gut microbiota, and CVD pathogenesis, and are further
confirmed by cecal as well as microbial transplant studies that demonstrated transmissibility
of microbiota-specific effects that satisfied Koch’s postulate71. Specifically in CKD, fecal
transplant experiments demonstrated that transfer of microbiota from CKD patients to
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antibiotic-treated mice produced elevated plasma TMAO with choline-feeding and

alterations in the TMAO metabolism functional profile72. Recently, a targeted competitive
inhibitor of TMA lyase was able to reduce both circulating TMAO levels and atherosclerosis
progression73. Recently, simultaneous publications of meta-analysis from three different
groups had largely confirmed that elevated TMAO levels is associated with increased CVD
risk and mortality74-76.

There have been major efforts to unravel the mechanistic underpinnings of adverse effects of
TMAO. Original studies implicated TMAO in alterations of cholesterol metabolism with
upregulation of scavenger receptors of macrophages64, 70. Several recent studies have
expanded the effects of TMAO exposure on promoting platelet hyperresponsiveness, as well
as robust inflammatory responses via the NF-kB pathway and inflammasome activation
from endothelial and vascular smooth muscle cells77-79. Interestingly, the TMAO precursor
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TMA is known to interact with the olfactory receptor Trace Amine-Associated Receptor 5
(TAAR5), a G-protein coupled receptor, even though TAAR5 does not recognize TMAO80.
Further investigations are warrant in identifying a potential receptor-mediated mechanism
for TMAO that would greatly advance the field.

TMAO is a predominant kidney-cleared metabolite. In the context of CKD, reduced renal

clearance unsurprisingly results in accumulation of TMAO, while renal transplantation

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results in dramatic reduction in plasma TMAO levels81, 82. Several reports have investigated
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the renal impact and prognostic value of TMAO in the CKD population (Table 2). Tang et al.
first showed that elevated TMAO in patients with reduced eGFR was prognostic for poor
outcomes4. The authors also demonstrated that choline and TMAO-fed mice directly
promoted renal fibrosis and was associated with increased phospho-Smad3 (a TGFβ-
signaling pathway), as well as markers of renal dysfunction such as cystatin C and kidney
injury marker-14. Subsequent studies have observed that in those with estimated glomerular
filtration rate <60 ml/min/1.73m2 TMAO levels were associated with atherosclerotic
burden82. There also appears to be racial differences in the prognostic potential of TMAO
with white patients at greater risk than black patients, which interestingly falls in line with
the dialysis vintage disparity between black and white patients83, 84. A recent study has even
indicated that TMAO may confer stronger prognostic value in patients with any degree of
renal dysfunction85.
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Despite the numerous clinical associations reported for TMAO in CKD patients,
mechanisms for TMAO excretion has been scant to date. Recently, a specific renal
transporter for TMAO has been suggested by several in vitro studies86, 87. These studies
identified organic cation transporter 2 (OCT2), a well-studied xenobiotic transporter as
being involved in TMAO excretion. In kinetic experiments, the Km of OCT2 for TMAO has
been reported to range from 7mM to 72mM. Unfortunately, given that the average
concentration of TMAO in the general population is only in the micromolar range (a 1,000×
fold difference), the possibility of OCT2 being a physiologically important transporter is less
plausible69. Although OCT1/2 knockout mice exhibited slightly elevated TMAO levels, no
OCT2 polymorphisms were associated with changes in plasma TMAO86, 87. Furthermore,
introduction of TMAO at the levels observed in uremia did not alter the transport of
metformin, a prominent OCT2 substrate88.
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From clinical observations, dialytic clearance data used to calculate the differences in
TMAO and creatinine clearance found that those with CKD appeared to have reduced
TMAO clearance capacities89. Moreover, in healthy individuals with normal renal function,
they appeared to have increased TMAO clearances above that of creatinine, suggesting that
the kidneys actively secrete TMAO. In another experiment, TMAO was identified as one of
16 metabolites that were associated with the development of CKD in prospective follow-
up39. While venous-arterial concentration ratio of choline was less than creatinine, that of
TMAO appeared to be similar to creatinine in a series of patients with CKD, suggesting that
those with reduced kidney function also appeared to have a loss of TMAO secretory
function.
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Therapeutic Options Targeting Gut Microbiota

Multiple approaches are taken in therapy which ranges from direct modification of diet,
prebiotics, probiotics, synbiotics, toxin-absorbing products, and finally, novel advances in
the field suggest the potential for targeted interventions through bacterial enzyme inhibition.
Here, we do not focus on specific microbial species, as significant discrepancies are reported
in the literature. Even bacteria amongst the same taxa may contain different functional

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capacities. However, expansion of new technology such as metagenomics will be able to

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better distinguish this varying functional capacity of our microbiota.

Dietary Modification
Dietary exposure may be one of the biggest contributors towards balancing the intestinal
microbiota composition. Vegetarian diets in normal populations have decreased
concentrations and generating capacities of protein-bound uremic solutes such as pCS and
IS44, 90. Though a low-protein diet is often recommended in dietary guidelines for CKD
patients, large studies such as the Modification of Diet in Renal Disease has found little
benefit in preventing CKD progression91, 92. In a study involving 40 patients with moderate-
to-advanced CKD, a lower ratio of dietary fiber to protein was associated with higher blood
levels of IS and pCS93. Although interventions in dietary changes such as excluding dietary
toxin sources have been attempted with promising results94, 95, it is a major lifestyle change
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and can be very difficult to adhere to. It is important to note that “heart healthy” diets are
often high in dietary fiber that may lead to the production of protective SCFAs largely come
from sources such as vegetables and fruits43, 47. Unfortunately, this type of diet also contains
high levels of potassium and phosphorus, which when faced with poor clearance in CKD
patients, can be associated with increased mortality risk96. This is an area that would benefit
from more rigorous and scalable interventions even with unconventional diets and taking
into account of gut microbial compositions96, 97.

Prebiotics, Probiotics, and Synbiotics

Prebiotics are indigestible compounds which can improve the microbiota composition and
subsequently, the functional capacity98. Probiotics then are living organisms that can be
consumed99. Finally, synbiotics contain a mixture of these two products. The findings from
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these studies have been positive but inconsistent, and often only a partial set of “important”
uremic toxins were shown to be decreased. Administration of prebiotics such as inulin or
resistant starch was shown to decrease pCS and IS, respectively100, 101, while decrease in IS
was observed when given a lactic acid bacteria preparation IS102 or Bifidobacterium in a
gastric acid-resistant pill102. Examples of synbiotics include work from Nakabayashi et al.
where a preparation combining Lactobacillus, Bifidobacterium, and galacto-
oligosaccharides resulted in decreased pCS103. In the SYNERGY trial, CKD patients
received 15g/day of a mixture of fermentable dietary fibers and probiotic strains from the
Bifidobacterium, Lactobacillus, and Streptococcus genera also observed reduction in pCS
but not IS104.
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Absorption Therapy
A prominent example of adsorption therapy is the oral activated charcoal adsorbent,
AST-120. This polymer has been shown in preclinical studies to restore colonic tight
junction proteins as well as decreasing plasma endotoxin and inflammatory markers105.
These therapies had strong preclinical data that supported its ability to reduce IS and pCS
levels. However in randomized controlled trials, AST-120 did not yield beneficial outcomes,
largely due to being underpowered by lower event rates106.

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Over the past years, the number of known uremic solutes has steadily increased to over
15041. It is clear that no single factor can be held responsible for the progression of these
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complex trait diseases, and different metabolites may have different characteristics. As an
example, TMAO has demonstrated unique traits which differ from that of IS and pCS which
can help to serve as another model microbial metabolite in the development of biomarkers
for diagnostic and prognostic purposes. It is not protein bound like IS and pCS and yet is
still poorly removed during hemodialysis89. This occurs in part due to decreased volume of
distribution as well as the more efficient excretion of TMAO by the kidney compared to the
dialytic standard urea. Observations from Aranov et al. have shown that colectomy does not
alter levels of monomethyl- and dimethylarginine levels compared to that of IS and pCS37.
Curiously, the AST-120 studies in nephrectomized rats also reveal significant increases in
TMAO levels despites decreases in IS and pCS107. This evidence illustrates the need for
future studies towards uremic toxin removal to be improved through experimental rigor and
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a systematic approach.

Targeted Inhibition of Microbial Enzymes

Along with the ideas of absorbing excess toxins, targeted reduction of uremic toxin
production has been demonstrated for TMAO with robust preclinical data. The small
molecule mimetic of choline, 3,3-dimethyl-1-butanol (DMB), is a potent antagonist against a
wide array of TMA lyases without affecting their survival73. Wang et al. demonstrated that
DMB feeding along with TMAO showed in non-lethal inhibition of TMA generation.
Specifically, choline-fed ApoE−/− mice treated with DMB demonstrated decreased plasma
TMAO levels along with attenuated atherosclerosis development compared to no DMB
treatment73. This finding extends previous work with antibiotics for targeted elimination of
TMAO generation, and may have a lower likelihood of developing “resistance” than
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antibiotics. Interestingly, DMB is also found naturally in foods such as extra-virgin olive oil,
which is a prominent component of the Mediterranean diet.

Meanwhile, studies have identified various compounds being able to modify TMAO levels,
including aspirin108, resveratrol109, and meldonium (an analog of γ-butyrobetaine)110. The
exact mechanisms are largely unknown. Additional validation studies will be important to
assess the potential utility of these treatments.

Conclusion
Gut microbiota is now recognized as an important contributor to health that is easily
perturbed not only by host factors but also by lifestyle and environmental changes. Gut
microbiota can stimulate the immune system to cause inflammation, and have profound
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impact on the host by metabolizing dietary precursors and generate metabolites with both
beneficial and toxic paracrine and endocrine effects. Large scale – omics technology will
now enable us to better quantify the litany of circulating metabolites to identify novel
patterns to diagnose, predict, and treat disease. However, systematic approaches are
necessary to study this subject and beyond just knowing that these microbial metabolites are
present, but also understanding their mechanistic contributions. Rigorous methodology
along with the state-of-the art technologies will pave way for novel therapeutic discoveries

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 Li and Wilson Tang Page 11

for CKD patients to prevent CVD as we continue to explore and develop our understanding
Author Manuscript

in this uncharted territory.

Acknowledgments
Mr. Li is a recipient of the 2016 Sarnoff Cardiovascular Fellowship Award. Dr. Tang is supported by grant funding
from the National Institutes of Health (R01DK106000, R01HL126827).

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Concentration, Disease Progression, and Mortality in CKD Patients. PLoS One. 2016;
11:e0161074. [PubMed: 27513517]
131. Li DY, Tang WHW. Gut Microbiota and Atherosclerosis. Curr Atheroscler Rep. 2017; 19:39.
[PubMed: 28842845]
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Figure 1.
Dietary precursors are modified by the microbiota to generate an array of uremic toxins
through multiple mechanisms at the level of the microbiota, intestinal wall, and liver. Influx
of uremic toxins results in microbiota imbalance along with bacterial translocation and
inflammation. These factors contribute to the multitude of systemic effects covered in this
review.
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Figure 2.
Overview of TMAO metabolism. Dietary precursors such as red meat, fish, and eggs contain
compounds that contain the TMA moiety which is released as TMA, absorbed into
circulation and converted to TMAO in the liver. Circulating TMAO has been implicated in
processes such as atherosclerosis, platelet activation as well as vascular inflammation. These
processes contribute to the development of renal dysfunction. Destruction of tubular
secretion mechanisms result in the accumulation of TMAO in the disease setting and
contribute to a feed-forward cycle. Modified from Tomlinson et al. 2017.
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Table 1

Summary of Mechanistic Studies on Indoxyl Sulfate and P-Cresyl Sulfate Effect on the Heart and Kidney
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Author Cell/Organ System Uremic Toxin Main Observed Effect Molecular Mediator Implicated
Dou 2007111 Endothelium IS Induction ROS, inhibition glutathione NADPH Oxidase ↑

Ito 2010112 Endothelium IS Increased endothelial/leukocyte interaction NADPH oxidase; E-selectin ↑

Lekawanvijit 2010113 Cardiac Fibroblast IS Cardiac fibrosis and inflammation Cytokines and Mitogen-activated
protein kinase: MEK 1/2; p38;
Nuclear Factor-kB ↑
Bolati 2011 Tubular Cell IS Increased epithelial to mesenchymal E-cadherin ↓
transition
Sun 2012114 Proximal Tubular Cell IS, PCS Increased cytokine and inflammatory gene a-Smooth muscle actin; Smad2/3
expression and Smad4 ↓
Sun 2012115 Proximal Tubular Cell IS, PCS Increased Klotho gene methylation and Klotho ↓
renal fibrosis
Koppe 2013116 Myotube, Myocyte PCS Increased insulin resistance, increased Phosphoinositide 3-kinase;
lipolysis, decreased lipogenesis Insulin receptor substrate-1;
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Extracellular-regulated kinase
1/2 ↓
Chitalia 2013117 Smooth Muscle Cells IS Increased tissue factor generation Tissue factor ↑

Yisireyili 2013118 Cardiac Tissue IS Increased cardiac hypertrophy, fibrosis, Nuclear Factor (erythroid-derived
and oxidative stress 2) - like 2; Heme-
oxygenase-1119↓
Transforming Growth Factor-b ↑
Bolati 2013120 Tubular Cell IS Increased oxidative stress Nuclear Factor (erythroid-derived
2) - like 2; Heme-oxygenase-1 ↓
Sun 2014121 Proximal tubular cell IS, PCS EGF receptor activation EGF receptor downstream
signaling ↑
Han 2015122 Cardiac tissue PCS Production of reactive oxygen species NADPH oxidase ↑

Hung 2016123 Endothelial progenitor cells IS Impaired neovascularization HIF-1α and interleukin-10/
STAT3 ↓
Yang 2017124 Platelet IS Induce platelet hyperreactivity ROS/p38MAPK ↑
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Selected studies include those rated as “Strong” in review from Vanholder et al. 2014 along with selected publications since 2014.

Note – P-cresol, the precursor to PCS was originally thought to be the retention solute due to an unforeseen preparation artifact125. It was only the
early 2000s when PCS was identified and its biologic toxicity sought out.

Abbreviations: IS – Indoxyl Sulfate; PCS – P-Cresyl Sulfate

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Table 2

Summary of Clinical Investigations Between TMAO and Cardiovascular Related Outcomes in Various Patient Cohorts

Study Cohort Author Subjects Study Population Criteria Endpoint of Interest Results
Chronic Kidney Disease Tang et al.4 521 Stable subjects with CKD (estimated All-cause mortality over 5 years of HR 1.93, CI 1.13–3.29, P<0.05
glomerular filtration rate, <60 mL/min per highest quartile TMAO
1.73 m2)
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Stubbs et al.82 220 Individuals with CKD defined as eGFR<60 Atherosclerotic burden by modified TMAO to Gensini score
ml/min per 1.73 m2 Gensini Score; 4 year mortality per 10 correlation
μM TMAO r=0.17, P=0.02;
HR 1.26, CI 1.13 – 1.40,
P<0.001
Shafi et al.84 1,232 Mixed ethnicity cohort (35% white, 65% Composite of death by coronary event, HR, 3.89; 95% CI, 2.47 to
black) with available serum from the HEMO arrhythmia, sudden cardiac death, 6.13; P<0.001;
study group126 randomized clinical trial congestive heart failure, or HR 1.92, CI 1.28–2.86,
comparing standard or high dialyzer urea cerebrovascular; any-cause death over P=0.001
clearance. years of highest quintile TMAO
comparing white vs black patients
Kim et al.127 2,529 CKD patients with Stage 3b and 4 CKD, Time to myocardial infarction, unstable HR 1.23, CI 1.06–1.42, P =
under nephrology care, in urban and rural angina, ischemic stroke, coronary 0.0059
centers across Canada. revascularization, new onset coronary
heart disease, amputation, peripheral
artery bypass, and gangrene over 3 years
per ln TMAO
Missailidis et al.128 179 CKD 3–4 and CKD 5 patients classified All-cause mortality over 5 years of HR 4.32, CI 1.32–14.2, P =
according to eGFR combined mid and high tertile TMAO 0.016
Kaysen et al.129 Cardiovascular Adults with end-stage renal disease who Time to death; Time to cardiovascular HR 0.84, CI 0.65–1.09 P =
mortality = 152 initiated hemodialysis or peritoneal dialysis death or hospitalization per unit log 0.19;
for; All-cause TMAO HR 0.88, CI 0.57–1.35 P =
mortality = 235 0.55
RobinsonCohen et al.130 339 Adults with eGFR ≤ 90 ml/min/1.73m2 or a FMO3 variants at amino acid 158 and E/K heterozygous:
urinary protein to creatinine ratio of > 30 association with and mortality after 3.3 HR 1.97, CI 0.85–4.60;
mg/g years median follow-up K/K Homozygous:
2.22-fold, CI 0.89–5.48;

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additive model P = 0.04
Gruppen et al.85 5,469 Individuals age 28 to 75 from the Prevention Highest vs lowest quartile TMAO all- HR 1.18, CI 1.02–1.36 P =
of Renal and Vascular End-Stage Disease cause mortality in subjects with eGFR 0.023
(PREVEND) study with measured TMAO <90 mL/min/1.73 m2
values stratified by renal function
Meta-analysis Heianza et al.76 19,256 PubMed and Embase, 16 publications Associations between TMAO with the RR1.62; CI 1.45–1.80;
development of MACE or death I2=23.5%
Schiattarella et al.74 15,662; 13,944 MEDLINE, ISI Web of Science and Associations between TMAO plasma HR 1.91, CI 1.40–2.61, P <
SCOPUS, 17 publications levels with all-cause mortality; 0.0001, I2 = 94%;
MACCE; dose response HR 1.67, CI 1.33–2.11, P <
0.00001, I2 = 46%;
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Study Cohort Author Subjects Study Population Criteria Endpoint of Interest Results
RR per each 10 μmol/L
increment of TMAO 1.07, CI
1.04–1.11, P < 0.0001
Qi et al.75 8,139; 7,716 PubMed and Embase, 11 publications Association between TMAO plasma HR 1.23, CI: 1.07–1.42, I2 =
levels with cardiovascular events; all- 31.4%;
cause mortality HR 1.55, CI: 1.19–2.02, I2 =
80.8%
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Updated from Li et al. 2017131

Abbreviations: CI – 95% Confidence Interval; CKD – Chronic Kidney Disease; eGFR – estimated Glomerular Filtration Rate; HR - Hazard Ratio; MACE – Major Adverse Cardiovascular Events; MACCE
- Major Adverse Cardiac and Cerebrovascular Events; RR – Risk Ratio

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